Minoru Miyakoshi

Environmental temperature and cataract progression in experimental rat cataract models.

PURPOSE To clarify whether or not ambient temperature relates to cataract development or the progression of cataract formation. MATERIALS AND METHODS 36 Brown Norway rats were divided into two groups, a high-temperature (35 +/- 2 degrees C, H = high) breeding group and a regular-temperature (24 +/- 2 degrees C, L = low) group. Each group was further divided into an experimentally induced diabetic cataract subgroup (50 mg/kg streptozotocin, DM), an ultraviolet B exposure-induced cataract subgroup (200 mJ/cm2, UV), and a normal control subgroup (C = control). Slit-lamp microscopy and an anterior image analysis system (EAS-1000) were used to evaluate lens changes. RESULTS Both the HC and HUV groups in the 35 degrees C conditions showed higher light scattering than that of the 24 degrees C conditions (LC and LUV) 3 weeks after the start of the experiment. Nine weeks after the start of the experiment, all the rats of the UV subgroups (HUV and LUV) developed anterior subcapsular cataract. The temperature did not have much influence on the progression of the UV-B-induced cataract. From 18 days after the start of the experiment, the HC subgroup showed a wider light scattering area than the LC. An increase in abnormal nuclear scattering light in the crystalline lens of group HC was found in 9 weeks after the start of the experiment, and at the end of the experiment (78 weeks later), dense abnormal nuclear light scattering was found including the prenuclear area. In contrast, the HDM group in the 35 degrees C conditions showed slower cataract progression than that of the LDM group at 24 degrees C room temperature. CONCLUSIONS Although further experiments are necessary before we can draw any conclusions about temperature and nuclear changes, paying attention to the effects of temperature on the lens is worthwhile.

Cell Growth Inhibition and DNA Incorporation of Mitomycin C in Cell Culture

The present study was performed to clarify the effects of a 4-min exposure of mitomycin C (MMC) on cell growth, the cell cycle and MMC dose incorporated into DNA, using Chang’s cultured human conjunctival cells. A low dose of MMC ranging from 0.00025 to 0.004% showed dose-dependent cytotoxicity when cell growth was active. Fifty percent cell viability was found when cells were treated with 0.001% MMC. A flow cytometer showed that 0.001% MMC inhibited the DNA synthetic phase. After 0.04% MMC was exposed to 3 ×106 cells and immediately rinsed, DNA was isolated to measure the dose of MMC detected from DNA. The total amount of DNA was 7 µg from which 3 µg of MMC was detected by high performance liquid chromatography. The above results revealed that the lowest concentration of MMC which caused 50% cell viability and cell cycle inhibition was 0.001% and that MMC was rapidly incorporated into DNA.

Bile Acid Load on the DNA Distribution Pattern of Bile Ductules and Cholangiocarcinoma Induced by Diisopropanolnitrosamine in Hamsters

This study evaluated the influence of bile acid load on the DNA distribution pattern of proliferated bile ductules and cholangiocarcinoma induced by diisopropanolnitrosamine. Ninety hamsters were separated into control, tauro- and deoxycholic acid (DCA) groups. The DNA distribution pattern of intrahepatic lesions at 15–25 weeks was measured by cytofluorometry and classified into three types: I (-A, -B), II and III, according to the degree of dispersion on the DNA histogram. Regarding proliferated bile ductule lesions, all groups showed an increase in cell populations, indicating the dispersion of nuclear DNA content from the 4C to 6C ranges over the course of 25 weeks, and two groups with bile acids, especially the DCA group, revealed significant high incidences of lesions with type I-B plus II compared with those in the control group (p < 0.05, 0.01). Changes in carcinoma types were similar to those of bile ductule lesions, and the tumors in the DCA group had a significant high frequency of type II plus III (p < 0.05). In addition, heterogeneity of the DNA distribution pattern was observed within individual lesions of not only carcinoma but also bile ductules. These results suggest that bile acid load, especially DCA, promotes an increase in nuclear DNA content or DNA polyploidization and enhances the distribution of the DNA pattern of proliferating bile ductules and carcinoma. Furthermore, a bile ductule-carcinoma sequence may be present in the development of cholangiocarcinoma.

Adsorbability to and desorbability from Sephadex G-15 of sodium and phosphate ions

Sephadex G-15 is a well known gel having a molecular sieving function. This report describes the results of quantitative absorption experiments using sodium-22 labelled sodium chloride and phosphorus-32 labelled phosphoric acid

Exchange of the cation and anion of the sample (sodium or potassium chloride) with the cation and anion of the eluent (sodium or potassium phosphate buffer) and their elution, from a Sephadex G-15 column, in separate fractions

Abstract Various concentrations of sodium and potassium chloride were eluted with sodium or potassium phosphate buffer (0.025 M , pH 7.0) in various sample—buffer combinations from a Sephadex G-15 column. A more acidic buffer (pH 6.0) or a more concentrated buffer (0.125 M ) were also used as the eluent. By observing the elution behaviour of all ions in the eluate, it was found that the cation from the sample accompanied by phosphate ion from the eluent was eluted in earlier fractions, and the chloride ion from the sample accompanied by the eluents cation was eluted in later fractions in all homocationic and heterocationic sample–eluent combinations employed. The mechanism assumed was that chloride ion from the sample was eluted slowly and the phosphate ion from the eluent rapidly, resulting in the occurrence of cation-exchange reactions between the sample and the eluent.

Differential elution from a Sephadex G-15 column of sodium and phosphate ions of sodium phosphate with sodium or potassium phosphate buffer

Abstract When a sample solution containing sodium-22-labelled sodium chloride and carrier-free phosphorus-32-labelled phosphoric acid was eluted from a Sephadex G-15 column with either 0.025 M sodium or potassium phosphate buffer (pH 7.0), the labelled phosphate ion was eluted earlier than the sodium-22. The presence of cold 0.72 M sodium chloride with the sodium-22-labelled sodium chloride in the sample did not affect the elution sequence. When 1 M monosodium phosphate was eluted with distilled water from fresh and phosphate-treated Sephadex columns, the sodium and phosphate ions were eluted together in approximately the same fractions in both instances. From these observations, it is concluded that sodium ion repeatedly exchanges its partner phosphate ion with that in the eluent during its elution from Sephadex.

Potassium and sodium chloride ion pairs are presumed to constitute a complex during elution from a Sephadex G-15 column with sodium phosphate buffer.

When a mixed solution of 0.72 M potassium and sodium chloride was eluted from a Sephadex G-15 column with 0.025 M sodium phosphate buffer (pH 7.0), the elution profiles of ions showed that the potassium and chloride ion pair from the sample and the sodium and chloride ion pair produced by ion-exchange reaction, were eluted in the same fractions as if they constituted a complex. When a mixed solution of different concentrations of potassium and sodium chloride was eluted with the same buffer, the excess amount of one ion pair over the other was eluted freely from the presumed complex.

Differential elution of sodium or potassium dihydrogen- and hydrogenphosphate ions from a sephadex G-15 column with sodium or potassium chloride solution.

When a mixed solution of sodium or potassium dihydrogenphosphate and disodium or dipotassium hydrogenphosphate was eluted from a Sephadex G-15 column with either a sodium or potassium chloride solution, the elution profiles of ions showed that the hydrogenphosphate ion was eluted more rapidly than the dihydrogenphosphate ion. When the sample solutions containing potassium dihydrogenphosphate and/or dipotassium hydrogenphosphate, all of which were supplemented with phosphorus-32-labelled potassium dihydrogenphosphate, were eluted with sodium chloride solution, the elution profiles of radioactivity showed that the dihydrogenphosphate ion changed to hydrogenphosphate ion and vice versa, depending on the pH values of the sample solution and the availability of the cation of the eluent during elution for the phosphate ion to pair with.

Cytotoxic Effects of Antifungal Drugs on Cultured Human Conjunctival Cells

We evaluated the cytotoxicity of 3 antifungal drugs using cultured human conjunctival cells. Cell growth curve, electron microscopic appearance and analysis of DNA histograms were studied after 2 to 4 min exposures to the test solution. Amphotericin B inhibited cell growth dose-dependently. Amphotericin B from 50 µg/ml to 100 µg/ml revealed loss of microvilli and a remarkable decrease in cell population. Miconazole (10 mg/ml) destroyed the cells completely. Fluconazole, at a concentration usually used (1 mg/ml), did not cause inhibition of cell growth or damage to the cell surface. None of the drugs studied resulted in abnormal pattern on DNA histograms.